Conjugates of an anti-TNF-α antibody

ABSTRACT

Conjugates of an anti-TNF antibody and one or more nonpeptidic water soluble polymers are provided. Typically, the nonpeptidic water soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are compositions comprising conjugates, methods of making conjugates, and methods of administering compositions to a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC §371 application of InternationalApplication No. PCT/US2007/008738 filed Apr. 6, 2007, designating theUnited States, which claims priority to U.S. Application No. 60/790,339filed Apr. 7, 2006, both of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

Among other things, one or more embodiments of the present inventionrelate generally to conjugates comprising an anti-TNFα antibody (e.g.,an antibody having the ability to bind to tumor necrosis factor-alpha or“TNFα”) and a polymer. In addition, the invention relates to (amongother things) compositions comprising conjugates, methods forsynthesizing conjugates, and methods of administering a composition.

BACKGROUND OF THE INVENTION

Tumor necrosis factor-alpha (“TNFα), alternatively referred to as“cachexin” or “cachectin,” is a 185 amino acid-long cytokine that isreleased by damaged white blood cells, endothelium cells and certaintissues. TNFα is formed in vivo by the cleavage of a 212 amino acid-longprecursor transmembrane protein. Upon cleavage of this precursortransmembrane protein, soluble molecules are released that aggregate toform complexes. These complexes then bind to tumor necrosis factorreceptors (TNF-R) found on a variety of cells to thereby result in anarray of pro-inflammatory effects, such as the release of thepro-inflammatory cytokines interleukin-6 and interleukin-8, theenhancement of endothelial layer permeability (thereby allowing forleukocyte migration), the activation of neutrophils and eosinophils, andthe induction of tissue-degrading enzymes produced by synoviocytes andchondrocytes.

Elevated levels of TNFα are associated with many disease states. Forexample, increased concentrations of TNFα are often found in the jointsof individuals suffering from rheumatoid arthritis. In these patients,the induction of tissue-degrading enzymes by TNFα causes degradation anderosion of joint and bone tissues. In addition to rheumatoid arthritis,Crohn's disease is another disease associated with increasedconcentrations of TNFα. While the exact cause of Crohn's disease isunknown, patients suffering from Crohn's disease experience inflammationand ulceration of the digestive tract. Other diseases and conditionsthat have been linked to increased levels of TNFα include psoriaticarthritis, ulcerative colitis, plaque psoriasis, sarcoidosis, ankylosisspondylitis, and cytokine-induced islet destruction in autoimmunediabetes.

Current approaches for treating individuals suffering from rheumatoidarthritis (as well as other diseases associated with increased TNFα)include neutralizing or otherwise diminishing the ability of TNFα tobind to TNFα receptors in the body. In one such approach, patients areadministered monoclonal antibodies that bind to TNFα (i.e.,anti-TNFα-antibodies), thereby inhibiting TNFα's ability to bind to TNFαreceptors. Commercially available forms of anti-TNFα-antibodies areavailable, including, infliximab (marketed under the REMICADE® name,Centocor, Inc., Malvern, Pa.) and adalimumab (marketed under the HUMIRA™name, Abbott Laboratories, Abbott Park, Ill.). Infliximab is typicallyadministered over at least two hours via an intravenous infusion whileadalimumab is typically administered subcutaneously every two weeks.Because infliximab is a chimeric antibody, there is concern thatadministration of this antibody to humans can result in an immunogenicreaction. Further, even though adalimumab is a human monoclonal antibodyspecific for TNF, approximately 5% of adult rheumatoid arthritispatients developed low-titer antibodies to adalimumab at least onceduring treatment (as demonstrated over three studies) and the long termimmunogenicity of adalimumab is unknown.

Another approach for neutralizing or diminishing the effects of TNFαincludes binding circulating TNFα, thereby reducing the amount of TNFαavailable for binding to functioning cell surface receptors. Thisapproach can be effected by administering TNFα receptors (or TNFα-likereceptors). By administering an excess of exogenous TNFα receptors (orTNFα-like receptors), circulating TNFα is bound to the exogenous andnon-functioning receptors resulting in significantly decreased amountsof TNFα available for activating endogenous TNFα receptors. Commerciallyavailable pharmaceutical formulations that are based on this approachinclude etanercept (marketed under the ENBREL®, Immuunex Corporation,Thousand Oaks, Calif.), a p75 type II TNF soluble receptor. Although notcurrently available commercially, PEGsunercept (or PEG-sTNF-RI) is aPEGylated version of a p55 type I TNF receptor. It has been alleged thatetanercept has been associated with rare cases of central nervous systemdisorders such as multiple sclerosis, myelitis and optic neuritis andpancytopenia, including aplastic anemia. There is relatively littleexperience with PEGsunercept to know whether it will suffer from thesame concerns as etanercept.

Thus, there remains a need to address, for example, the immunogenicityconcerns associated with therapies intended to decrease the effects TNFαin vivo. The present invention is intended to address the immunogencityconcerns (and/or other concerns) by, for example, attaching awater-soluble polymer to an anti-TNF antibody, thereby forming aconjugate between the water-soluble polymer and the anti-TNF antibody.The present invention includes this and other embodiments, which arebelieved to be new and completely unsuggested by the art.

SUMMARY OF THE INVENTION

Accordingly, a conjugate is provided, the conjugate comprising ananti-TNFα antibody covalently attached, either directly or through aspacer moiety, to a nonpeptidic water-soluble polymer. The conjugate istypically provided as part of a composition.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an anti-TNFα antibody covalentlyattached through a hydrolytically stable linkage to a water-solublepolymer.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an anti-TNFα antibody covalentlyattached to a water-soluble polymer.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an anti-TNFα antibody covalentlyattached to a water-soluble polymer, wherein the anti-TNFα antibody iscovalently attached at an amine.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an anti-TNFα antibody covalentlyattached to a linear water-soluble polymer.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an anti-TNFα antibody covalentlyattached to a branched water-soluble polymer.

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is not a dimer or trimer (and therefore thecorresponding anti-TNFα antibody residue within the conjugate is not adimer or trimer).

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is monovalent (and therefore the correspondinganti-TNFα antibody residue within the conjugate is monovalent).

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is not a CDR-grafted (and therefore thecorresponding anti-TNFα antibody residue within the conjugate is notCDR-grafted).

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is a full length antibody (and therefore thecorresponding anti-TNFα antibody residue within the conjugate is a fulllength antibody).

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is not galactosylated (and therefore thecorresponding anti-TNFα antibody residue within the conjugate is not agalactosylated).

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is not glycosylated (and therefore thecorresponding anti-TNFα antibody residue within the conjugate is notglycosylated).

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein:

POLY is a water-soluble polymer;

(a) is either zero or one;

X, when present, is a spacer moiety comprised of one or more atoms;

R¹ is H or an organic radical containing 1 to 3 carbon atoms; and

ATA is a residue of an anti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein n ranges from about 3 to about 1400 and ATA is a residue of ananti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein n ranges from about 3 to about 1400 and ATA is a residue of ananti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein:

(n) is 2 to 4000;

(g′) is 0, 1, 2 or 3;

(c) is 1 to 10;

each R² is H or an organic radical;

each R³ is H or an organic radical;

(j) is 0 to 20; and

ATA is a residue of an anti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein:

(n) is 2 to 4000;

(g′) is 0, 1, 2 or 3;

(c) is 1 to 10;

each R² is H or an organic radical;

each R³ is H or an organic radical;

(j) is 0 to 20; and

ATA is a residue of an anti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein:

POLY is a water-soluble polymer;

(a) is either zero or one;

X, when present, is a spacer moiety comprised of one or more atoms; and

ATA is a residue of an anti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein:

POLY is a water-soluble polymer;

(a) is either zero or one;

X, when present, is a spacer moiety; and

ATA is a residue of an anti-TNFα antibody.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising the following structure:

wherein:

POLY is a water-soluble polymer;

(a) is either zero or one;

(j) is zero or an integer from 1 to about 20;

(b) is zero or an integer from 1 to about 10;

each R², when present, is H or an organic radical;

each R³, when present, Is H or an organic radical; and

ATA is a residue of an anti-TNFα antibody.

In one or more embodiments of the invention, the water-soluble polymerused to form the conjugate is poly(ethylene glycol). The weight averagemolecular weight of the water-soluble polymer can be within one or moreof the following ranges: from about 6,000 Daltons to about 100,000Daltons; from about 10,000 Daltons to about 85,000 Daltons; and fromabout 20,000 Daltons to about 65,000 Daltons.

In one or more embodiments of the invention, the anti-TNFα antibody usedto form the conjugate is either infliximab or adalimumab (and thereforethe corresponding anti-TNFα antibody residue located with the conjugateis either infliximab or adalimumab).

In one or more embodiments of the invention, a composition is provided,the composition comprising a plurality of conjugates, each conjugatecomprised of a residue of an anti-TNF antibody attached, either directlyor through a spacer moiety comprised of one or more atoms, to a PEGmolecule, wherein at least 50% of all conjugates in the composition areN-terminally monoPEGylated.

In one or more embodiments of the invention, a composition is provided,the composition comprising a plurality of conjugates, each conjugatecomprised of a residue of an anti-TNF antibody attached, either directlyor through a spacer moiety comprised of one or more atoms, to awater-soluble polymer, wherein at least 75% of all conjugates in thecomposition have a residue of an anti-TNF antibody attached, eitherdirectly or through a spacer moiety comprised of one or more atoms, tofive or fewer water-soluble polymers.

In one or more embodiments of the invention, a composition is provided,the composition comprising a plurality of conjugates, each conjugatecomprised of a residue of an anti-TNF antibody attached, either directlyor through a spacer moiety comprised of one or more atoms, to awater-soluble polymer, wherein at least 75% of all conjugates in thecomposition have a residue of an anti-TNF antibody attached, eitherdirectly or through a spacer moiety comprised of one or more atoms, tothree or fewer water-soluble polymers.

In one or more embodiments of the invention, a method for delivering aconjugate is provided, the method comprising the step of subcutaneouslyadministering to the patient a composition comprised of a conjugate of aresidue of an anti-TNF antibody and a water-soluble polymer.

DETAILED DESCRIPTION OF THE INVENTION

Before describing one or more embodiments of the present invention indetail, it is to be understood that this invention is not limited to theparticular polymers, synthetic techniques, anti-TNF antibodies, and thelike, as such may vary.

It must be noted that, as used in this specification and the intendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a polymer” includes a single polymer as well as two ormore of the same or different polymers, reference to “an optionalexcipient” refers to a single optional excipient as well as two or moreof the same or different optional excipients, and the like.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable and encompass any nonpeptidic water-solublepoly(ethylene oxide). Typically, PEGs for use in accordance with theinvention comprise the following structure “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” depending uponwhether or not the terminal oxygens have been displaced. Throughout thespecification and claims, it should be remembered that the term “PEG”includes structures having various terminal or “end capping” groups andso forth. The term “PEG” also means a polymer that contains a majority,that is to say, greater than 50%, of —OCH₂CH₂— repeating subunits. Withrespect to specific forms, the PEG can take any number of a variety ofmolecular weights, as well as structures or geometries such as“branched,” “linear,” “forked,” “multifunctional,” and the like, to bedescribed in greater detail below.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃O(CH₂CH₂O)_(n)— andCH₃(OCH₂CH₂)_(n)—]. In addition, saturated, unsaturated, substituted andunsubstituted forms of each of the foregoing are envisioned. Moreover,the end-capping group can also be a silane. The end-capping group canalso advantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) to which thepolymer is coupled can be determined by using a suitable detector. Suchlabels include, without limitation, fluorescers, chemiluminescers,moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions,radioactive moieties, and the like. Suitable detectors includephotometers, films, spectrometers, and the like. The end-capping groupcan also advantageously comprise a phospholipid. When the polymer has anend-capping group comprising a phospholipid, unique properties areimparted to the polymer and the resulting conjugate. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may, however, containone or more monomers or segments of monomers that are naturallyoccurring, so long as the overall polymer structure is not found innature.

The term “water soluble” as in a “water-soluble polymer” is any polymerthat is soluble in water at room temperature. Typically, a water-solublepolymer will transmit at least about 75%, more preferably at least about95%, of light transmitted by the same solution after filtering. On aweight basis, a water-soluble polymer will preferably be at least about35% (by weight) soluble in water, more preferably at least about 50% (byweight) soluble in water, still more preferably about 70% (by weight)soluble in water, and still more preferably about 85% (by weight)soluble in water. It is most preferred, however, that the water-solublepolymer is about 95% (by weight) soluble in water or completely solublein water.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number average molecular weight or a weightaverage molecular weight. Unless otherwise indicated, all references tomolecular weight herein refer to the weight average molecular weight.Both molecular weight determinations, number average and weight average,can be measured using gel permeation chromatography or other liquidchromatography techniques. Other methods for measuring molecular weightvalues can also be used, such as the use of end-group analysis or themeasurement of colligative properties (e.g., freezing-point depression,boiling-point elevation, or osmotic pressure) to determine numberaverage molecular weight or the use of light scattering techniques,ultracentrifugation or viscometry to determine weight average molecularweight. The polymers of the invention are typically polydisperse (i.e.,number average molecular weight and weight average molecular weight ofthe polymers are not equal), possessing low polydispersity values ofpreferably less than about 1.2, more preferably less than about 1.15,still more preferably less than about 1.10, yet still more preferablyless than about 1.05, and most preferably less than about 1.03.

The term “active” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “spacer moiety,” “linkage” and “linker” are used herein torefer to an atom or a collection of atoms optionally used to linkinterconnecting moieties such as a terminus of a polymer segment and ananti-TNF antibody or an electrophile or nucleophile of an anti-TNFantibody. The spacer moiety may be hydrolytically stable or may includea physiologically hydrolyzable or enzymatically degradable linkage.Unless the context clearly dictates otherwise, a spacer moietyoptionally exists between any two elements of a compound (e.g., theprovided conjugates comprising a residue of the anti-TNF antibody andwater-soluble polymer can attached directly or indirectly through aspacer moiety).

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering groups as a substituent. For substitutions on aphenyl ring, the substituents may be in any orientation (i.e., ortho,meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

An “organic radical” as used herein shall include alkyl, substitutedalkyl, aryl, substituted aryl,

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or with anelectrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient” or “carrier” refers to anexcipient that may optionally be included in the compositions of theinvention and that causes no significant adverse toxicological effectsto the patient. “Pharmacologically effective amount,” “physiologicallyeffective amount,” and “therapeutically effective amount” are usedinterchangeably herein to mean the amount of a polymer-anti-TNF antibodyconjugate that is needed to provide a desired level of the conjugate (orcorresponding unconjugated anti-TNF antibody) in the bloodstream or inthe target tissue. The precise amount will depend upon numerous factors,e.g., the particular anti-TNF antibody, the components and physicalcharacteristics of the therapeutic composition, intended patientpopulation, individual patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents of the invention willtypically contain from about 3-100 functional groups, or from 3-50functional groups, or from 3-25 functional groups, or from 3-15functional groups, or from 3 to 10 functional groups, or will contain 3,4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.

The term “anti-TNFα antibody” as used herein, refers to an a moiety(such as a full length antibody) which neutralizes the biologicalactivity of human TNFα through binding to human TNFα, thereby decreasingthe ability of the bound human TNFα bind to anti-TNFα receptors. Theanti-TNFα antibody will also have at least one electrophilic group ornucleophilic group suitable for reaction with a polymeric reagent. Inaddition, the term “anti-TNFα antibody” encompasses both the anti-TNFαantibody prior to conjugation as well as the anti-TNFα antibody residuefollowing conjugation. As will be explained in further detail below, oneof ordinary skill in the art can determine whether any given moiety isan anti-TNFα antibody. Exemplary anti-TNFα antibodies include infliximaband adalimumab.

The term “substantially homologous” means that a particular subjectsequence, for example, a mutant sequence, varies from a referencesequence by one or more substitutions, deletions, or additions, the neteffect of which does not result in an adverse functional dissimilaritybetween the reference and subject sequences. For purposes of the presentinvention, sequences having greater than 95 percent homology, equivalentbiological properties, and equivalent expression characteristics areconsidered substantially homologous. For purposes of determininghomology, truncation of the mature sequence should be disregarded.Sequences having lesser degrees of homology, comparable bioactivity, andequivalent expression characteristics are considered substantialequivalents.

The term “fragment” means any fragment of a full length anti-TNFαantibody that retains the ability to bind to TNFα.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of anactive agent (e.g., conjugate), and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” means nearly totally or completely, for instance,satisfying one or more of the following: greater than 50%, 51% orgreater, 75% or greater, 80% or greater, 90% or greater, and 95% orgreater of the condition.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is H is or H; Glutamine is Gln or Q; Asparagine is Asnor N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid isGlu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Argor R; and Glycine is Gly or G.

Unless the context clearly dictates otherwise, when the term “about”precedes a numerical value, the numerical value is understood tomean±10% of the stated numerical value.

Turning to one or more embodiments of the invention, a conjugate isprovided, the conjugate comprising an anti-TNFα antibody covalentlyattached, either directly or through a spacer moiety, to a nonpeptidicwater-soluble polymer. The conjugates of the invention will have one ormore of the following features.

The Anti-TNFα Antibody

As previously stated, the conjugate generically comprises an anti-TNFαantibody covalently attached, either directly or through a spacermoiety, to a nonpeptidic water-soluble polymer. As used herein, the term“anti-TNFα antibody” shall refer to the anti-TNFα antibody prior toconjugation as well as to the anti-TNFα antibody following attachment toa nonpeptidic water-soluble polymer. It will be understood, however,that when the original anti-TNFα antibody is attached to a nonpeptidicwater-soluble polymer, the anti-TNFα antibody is slightly altered due tothe presence of one or more covalent bonds associated with linkage tothe polymer optionally through a spacer moiety. Often, this slightlyaltered form of the anti-TNFα antibody attached to another molecule isreferred to a “residue” of the anti-TNFα antibody. The anti-TNFαantibody in the conjugate can be any peptide that provides anti-TNFαantibody activity.

The anti-TNFα antibody can be derived from conventional techniques forforming antibodies.

For any given antibody proposed to be an anti-TNFα antibody suitable foruse in the conjugates described herein, it is possible to determinewhether that moiety has anti-TNFα antibody activity. For example, it ispossible to adhere a composition comprising the proposed antibody tocolumn and pass labeled human TNFα through the column. Subsequentdetection of labels being retained on the column (as the result ofhaving been bound to the proposed antibody) indicates that the proposedantibody is suitable for use as an anti-TNFα antibody herein.

The Water-Soluble Polymer

As previously discussed, each conjugate comprises an anti-TNFα antibodyattached to a water-soluble polymer. With respect to the water-solublepolymer, the water-soluble polymer is nonpeptidic, nontoxic,non-naturally occurring and biocompatible. With respect tobiocompatibility, a substance is considered biocompatible if thebeneficial effects associated with use of the substance alone or withanother substance (e.g., an active agent such as an anti-TNF antibody)in connection with living tissues (e.g., administration to a patient)outweighs any deleterious effects as evaluated by a clinician, e.g., aphysician. With respect to non-immunogenicity, a substance is considerednonimmunogenic if the intended use of the substance in vivo does notproduce an undesired immune response (e.g., the formation of antibodies)or, if an immune response is produced, that such a response is notdeemed clinically significant or important as evaluated by a clinician.It is particularly preferred that the nonpeptidic water-soluble polymeris biocompatible and nonimmunogenic.

Further, the polymer is typically characterized as having from 2 toabout 300 termini. Examples of such polymers include, but are notlimited to, poly(alkylene glycols) such as polyethylene glycol (“PEG”),poly(propylene glycol) (“PPG”), copolymers of ethylene glycol andpropylene glycol and the like, poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and combinations of any of the foregoing.

The polymer is not limited to a particular structure and can be linear(e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g.,forked PEG or PEG attached to a polyol core), dendritic, or withdegradable linkages. Moreover, the internal structure of the polymer canbe organized in any number of different patterns and can be selectedfrom the group consisting of homopolymer, alternating copolymer, randomcopolymer, block copolymer, alternating tripolymer, random tripolymer,and block tripolymer.

Typically, activated PEG and other activated water-soluble polymers(i.e., polymeric reagents) are activated with a suitable activatinggroup appropriate for coupling to a desired site on the anti-TNFantibody. Thus, a polymeric reagent will possess a reactive group forreaction with the anti-TNF antibody. Representative polymeric reagentsand methods for conjugating these polymers to an active moiety are knownin the art and further described in Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Plenus Press, New York (1992), and inZalipsky (1995) Advanced Drug Reviews 16: 157-182.

Typically, the weight-average molecular weight of the water-solublepolymer in the conjugate is from about 100 Daltons to about 150,000Daltons. Exemplary ranges, however, include weight-average molecularweights in the range of greater than 5,000 Daltons to about 100,000Daltons, in the range of from about 6,000 Daltons to about 90,000Daltons, in the range of from about 10,000 Daltons to about 85,000Daltons, in the range of greater than 10,000 Daltons to about 85,000Daltons, in the range of from about 20,000 Daltons to about 85,000Daltons, in the range of from about 53,000 Daltons to about 85,000Daltons, in the range of from about 25,000 Daltons to about 120,000Daltons, in the range of from about 29,000 Daltons to about 120,000Daltons, in the range of from about 35,000 Daltons to about 120,000Daltons, and in the range of from about 40,000 Daltons to about 120,000Daltons. For any given water-soluble polymer, PEGs having a molecularweight in one or more of these ranges are preferred.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons,about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons,about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons,about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branchedversions of the water-soluble polymer (e.g., a branched 40,000 Daltonwater-soluble polymer comprised of two 20,000 Dalton polymers) having atotal molecular weight of any of the foregoing can also be used. In oneor more embodiments, the conjugate will not have any PEG moietiesattached, either directly or indirectly, with a PEG having a weightaverage molecular weight of less than about 6,000 Daltons.

When used as the polymer, PEGs will typically comprise a number of(OCH₂CH₂) monomers [or (CH₂CH₂O) monomers, depending on how the PEG isdefined]. As used throughout the description, the number of repeatingunits is identified by the subscript “n” in “(OCH₂CH₂)_(n).” Thus, thevalue of (n) typically falls within one or more of the following ranges:from 2 to about 3400, from about 100 to about 2300, from about 100 toabout 2270, from about 136 to about 2050, from about 225 to about 1930,from about 450 to about 1930, from about 1200 to about 1930, from about568 to about 2727, from about 660 to about 2730, from about 795 to about2730, from about 795 to about 2730, from about 909 to about 2730, andfrom about 1,200 to about 1,900. For any given polymer in which themolecular weight is known, it is possible to determine the number ofrepeating units (i.e., “n”) by dividing the total weight-averagemolecular weight of the polymer by the molecular weight of the repeatingmonomer.

One particularly preferred polymer for use in the invention is anend-capped polymer, that is, a polymer having at least one terminuscapped with a relatively inert group, such as a lower C₁₋₆ alkoxy group,although a hydroxyl group can also be used. When the polymer is PEG, forexample, it is preferred to use a methoxy-PEG (commonly referred to asmPEG), which is a linear form of PEG wherein one terminus of the polymeris a methoxy (—OCH₃) group, while the other terminus is a hydroxyl orother functional group that can be optionally chemically modified.

In one form useful in one or more embodiments of the present invention,free or unbound PEG is a linear polymer terminated at each end withhydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH,wherein (n) typically ranges from zero to about 4,000.

The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol can represent the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—,wherein (n) is as defined as above.

Another type of PEG useful in one or more embodiments of the presentinvention is methoxy-PEG-OH, or mPEG in brief, in which one terminus isthe relatively inert methoxy group, while the other terminus is ahydroxyl group. The structure of mPEG is given below.CH₃O—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OHwherein (n) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine. Depending on the specific anti-TNF antibody used, the reactiveester functional group of the disubstituted lysine may be furthermodified to form a functional group suitable for reaction with thetarget group within the anti-TNF antibody.

In addition, the PEG can comprise a forked PEG. An example of a forkedPEG is represented by the following structure:

wherein: X is a spacer moiety of one or more atoms and each Z is anactivated terminal group linked to CH by a chain of atoms of definedlength. International Application No. PCT/US99/05333, discloses variousforked PEG structures capable of use in one or more embodiments of thepresent invention. The chain of atoms linking the Z functional groups tothe branching carbon atom serve as a tethering group and may comprise,for example, alkyl chains, ether chains, ester chains, amide chains andcombinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymer,including any of the above-described polymers. For example, PEG can beprepared with ester linkages in the polymer that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include: carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol;orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, and a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by, for example,a phosphoramidite group, e.g., at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

Such optional features of the conjugate, i.e., the introduction of oneor more degradable linkages into the polymer chain, may provide foradditional control over the final desired pharmacological properties ofthe conjugate upon administration. For example, a large and relativelyinert conjugate (i.e., having one or more high molecular weight PEGchains attached thereto, for example, one or more PEG chains having amolecular weight greater than about 10,000, wherein the conjugatepossesses essentially no bioactivity) may be administered, which ishydrolyzed to generate a bioactive conjugate possessing a portion of theoriginal PEG chain. In this way, the properties of the conjugate can bemore effectively tailored to balance the bioactivity of the conjugateover time.

The water-soluble polymer associated with the conjugate can also be“cleavable.” That is, the water-soluble polymer cleaves (either throughhydrolysis, enzymatic processes, or otherwise), thereby resulting in theunconjugated anti-TNF antibody. In some instances, cleavable polymersdetach from the anti-TNF antibody in vivo without leaving any fragmentof the water-soluble polymer. In other instances, cleavable polymersdetach from the anti-TNF antibody in vivo leaving a relatively smallfragment (e.g., a succinate tag) from the water-soluble polymer. Anexemplary cleavable polymer includes one that attaches to the anti-TNFantibody via a carbonate linkage.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning nonpeptidic and water-soluble polymer is by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the term “polymeric reagent” generally refers to an entiremolecule, which can comprise a water-soluble polymer segment and afunctional group.

As described above, a conjugate of the invention comprises awater-soluble polymer covalently attached to an anti-TNFα antibody.Typically, for any given conjugate, there will be one to threewater-soluble polymers covalently attached to one or more moietieshaving anti-TNF antibody activity. In some instances, however, theconjugate may have 1, 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymersindividually attached to an anti-TNFα antibody.

The particular linkage within the anti-TNFα antibody and the polymerdepends on a number of factors. Such factors include, for example, theparticular linkage chemistry employed, the particular anti-TNFαantibody, the available functional groups within the anti-TNFα antibody(either for attachment to a polymer or conversion to a suitableattachment site), the presence of additional reactive functional groupswithin the anti-TNFα antibody, and the like.

Typically, a hydrolytically stable linkage, such as an amide, urethane(also known as carbamate), amine, thioether (also known as sulfide), orurea (also known as carbamide) linkage is employed as the linkage forcoupling the anti-TNFα antibody. Again, a preferred hydrolyticallystable linkage is an amide. In one approach, a water-soluble polymerbearing an activated ester can be reacted with an amine group on theanti-TNFα antibody to thereby result in an amide linkage.

For conjugates possessing a hydrolytically stable linkage that couplesthe anti-TNFα antibody to the polymer, the conjugate will typicallypossess a measurable degree of bioactivity. For instance, suchconjugates are typically characterized as having a bioactivitysatisfying one or more of the following percentages relative to that ofthe unconjugated anti-TNF antibody: at least about 2%, at least about5%, at least about 10%, at least about 15%, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, at least about 100%, and more than 105%(when measured in a suitable model, such as those well known in theart). Preferably, conjugates having a hydrolytically stable linkage(e.g., an amide linkage) will possess at least some degree of thebioactivity of the unmodified parent anti-TNFα antibody.

Amino groups on anti-TNFα antibody provide a point of attachment betweenthe anti-TNFα antibody and the water-soluble polymer. Lysine residues,each having an ε-amino acid that may be available for conjugation.Further, the N-terminal amine of anti-TNFα antibody can also serve as apoint of attachment.

There are a number of examples of suitable polymeric reagents useful forforming covalent linkages with available amines of an anti-TNFαantibody. Specific examples, along with the corresponding conjugate, areprovided in Table 1, below. In the table, the variable (n) representsthe number of repeating monomeric units and “—NH-ATA” represents theresidue of the anti-TNFα antibody following conjugation to the polymericreagent. While each polymeric portion [e.g., (OCH₂CH₂)_(n) or(CH₂CH₂O)_(n)] presented in Table 1 terminates in a “CH₃” group, othergroups (such as H and benzyl) can be substituted therefor.

TABLE 1 Amine-Specific Polymeric Reagents and the anti-TNFα antibodyConjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate

H₃C—(OCH₂CH₂)_(n)—O—CH₂CH₂—CH₂—NH-ATA Secondary Amine Linkage

H₃C—(OCH₂CH₂)_(n)—O—CH₂CH₂CH₂—CH₂—NH-ATA Secondary Amine Linkage

H₃C—(OCH₂CH₂)_(n)—O—CH₂CH₂—NH-ATA Secondary Amine Linkage

H₃CO—(CH₂CH₂O)_(n)—CH₂CH₂—NH-ATA Second Amine Linkage

Conjugation of a polymeric reagent to an amino group of an anti-TNFαantibody can be accomplished by a variety of techniques. In oneapproach, an anti-TNFα antibody can be conjugated to a polymeric reagentfunctionalized with a succinimidyl derivative (or other activated estergroup, wherein approaches similar to those described for thesealternative activated ester group-containing polymeric reagents can beused). In this approach, the polymer bearing a succinimidyl derivativecan be attached to the anti-TNFα antibody in an aqueous media at a pH of7 to 9.0, although using different reaction conditions (e.g., a lower pHsuch as 6 to 7, or different temperatures and/or less than 15° C.) canresult in the attachment of the polymer to a different location on theanti-TNFα antibody. In addition, an amide linkage can be formed reactingan amine-terminated nonpeptidic, water-soluble polymer with an anti-TNFαantibody bearing an activating a carboxylic acid group.

Typical of another approach useful for conjugating the anti-TNFαantibody to a polymeric reagent is use of reductive amination toconjugate a primary amine of an anti-TNFα antibody with a polymericreagent functionalized with a ketone, aldehyde or a hydrated formthereof (e.g., ketone hydrate, aldehyde hydrate). In this approach, theprimary amine from the anti-TNFα antibody reacts with the carbonyl groupof the aldehyde or ketone (or the corresponding hydroxyl-containinggroup of a hydrated aldehyde or ketone), thereby forming a Schiff base.The Schiff base, in turn, can then be reductively converted to a stableconjugate through use of a reducing agent such as sodium borohydride.Selective reactions (e.g., at the N-terminus are possible) are possible,particularly with a polymer functionalized with a ketone or analpha-methyl branched aldehyde and/or under specific reaction conditions(e.g., reduced pH).

Carboxyl groups represent another functional group that can serve as apoint of attachment on the anti-TNF antibody. Structurally, theconjugate will comprise the following:

where ATA and the adjacent carbonyl group corresponds to thecarboxyl-containing anti-TNFα antibody, X is a linkage, preferably aheteroatom selected from O, N(H), and S, and POLY is a water-solublepolymer such as PEG, optionally terminating in an end-capping moiety.

The C(O)—X linkage results from the reaction between a polymericderivative bearing a terminal functional group and a carboxyl-containinganti-TNFα antibody. As discussed above, the specific linkage will dependon the type of functional group utilized. If the polymer isend-functionalized or “activated” with a hydroxyl group, the resultinglinkage will be a carboxylic acid ester and X will be O. If the polymerbackbone is functionalized with a thiol group, the resulting linkagewill be a thioester and X will be S. When certain multi-arm, branched orforked polymers are employed, the C(O)X moiety, and in particular the Xmoiety, may be relatively more complex and may include a longer linkagestructure.

Water-soluble derivatives containing a hydrazide moiety are also usefulfor conjugation at a carbonyl. To the extent that the anti-TNFα antibodymoiety does not contain a carbonyl moiety, a carbonyl moiety can beintroduced by reducing any carboxylic acids (e.g., the C-terminalcarboxylic acid) and/or by providing glycosylated or glycated (whereinthe added sugars have a carbonyl moiety) versions of the anti-TNFαantibody. Specific examples of water-soluble derivatives containing ahydrazide moiety, along with the corresponding conjugates, are providedin Table 2, below. In addition, any water-soluble derivative containingan activated ester (e.g., a succinimidyl group) can be converted tocontain a hydrazide moiety by reacting the water-soluble polymerderivative containing the activated ester with hydrazine (NH₂—NH₂) ortert-butyl carbazate [NH₂NHCO₂C(CH₃)₃]. In the table, the variable (n)represents the number of repeating monomeric units and “═C-ATA”represents the residue of the anti-TNFα antibody following conjugationto the polymeric reagent. Optionally, the hydrazone linkage can bereduced using a suitable reducing agent. While each polymeric portion[e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented in Table 2 terminatesin a “CH₃” group, other groups (such as H and benzyl) can be substitutedtherefor.

TABLE 2 Carboxyl-Specific Polymeric Reagents and the anti-TNFα antibodyConjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate

Thiol groups contained within the anti-TNFα antibody can serve aseffective sites of attachment for the water-soluble polymer. Inparticular, cysteine residues provide thiol groups when the anti-TNFαantibody contains a cysteine. The thiol groups in such cysteine residuescan then be reacted with an activated PEG that is specific for reactionwith thiol groups, e.g., an N-maleimidyl polymer or other derivative, asdescribed in U.S. Pat. No. 5,739,208 and in International PatentPublication No. WO 01/62827.

Specific examples, along with the corresponding conjugate, are providedin Table 3, below. In the table, the variable (n) represents the numberof repeating monomeric units and “—S-ATA” represents the anti-TNFαantibody residue following conjugation to the water-soluble polymer.While each polymeric portion [e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)]presented in Table 3 terminates in a “CH₃” group, other groups (such asH and benzyl) can be substituted therefor.

TABLE 3 Thiol-Specific Polymeric Reagents and the anti-TNFα antibodyConjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate

H₃CO—(CH₂CH₂O)_(n)—CH₂CH₂CH₂CH₂—S—S•ATA Disulfide Linkage

ATA-S—S—CH₂CH₂—(CH₂CH₂O)_(n)—CH₂CH₂CH₂CH₂—S—S-ATA Disulfide Linkages

With respect to conjugates formed from water-soluble polymers bearingone or more maleimide functional groups (regardless of whether themaleimide reacts with an amine or thiol group on the anti-TNF antibody),the corresponding maleamic acid form(s) of the water-soluble polymer canalso react with the anti-TNFα antibody. Under certain conditions (e.g.,a pH of about 7-9 and in the presence of water), the maleimide ring will“open” to form the corresponding maleamic acid. The maleamic acid, inturn, can react with an amine or thiol group of an anti-TNFα antibody.Exemplary maleamic acid-based reactions are schematically shown below.POLY represents the water-soluble polymer, and ATA represents theanti-TNFα antibody.

A representative conjugate in accordance with the invention can have thefollowing structure:POLY-L_(0,1)-C(O)Z—Y—S—S-ATAwherein POLY is a water-soluble polymer, L is an optional linker, Z is aheteroatom selected from the group consisting of O, NH, and S, and Y isselected from the group consisting of C₂₋₁₀ alkyl, C₂₋₁₀ substitutedalkyl, aryl, and substituted aryl, and ATA is an anti-TNFα antibody.Polymeric reagents that can be reacted with an anti-TNFα antibody andresult in this type of conjugate are described in U.S. PatentApplication Publication No. 2005/0014903.

Conjugates can be formed using thiol-specific polymeric reagents in anumber of ways and the invention is not limited in this regard. Forexample, the anti-TNFα antibody—optionally in a suitable buffer(including amine-containing buffers, if desired)—is placed in an aqueousmedia at a pH of about 7-8 and the thiol-specific polymeric reagent isadded at a molar excess. The reaction is allowed to proceed for about0.5 to 2 hours, although reaction times of greater than 2 hours (e.g., 5hours, 10 hours, 12 hours, and 24 hours) can be useful if PEGylationyields are determined to be relatively low. Exemplary polymeric reagentsthat can be used in this approach are polymeric reagents bearing areactive group selected from the group consisting of maleimide, sulfone(e.g., vinyl sulfone), and thiol (e.g., functionalized thiols such as anortho pyridinyl or “OPSS”).

With respect to polymeric reagents, those described here and elsewherecan be purchased from commercial sources (e.g., Nektar Therapeutics,Huntsville, Ala.). In addition, methods for preparing the polymericreagents are described in the literature.

The attachment between the anti-TNFα antibody and the non-peptidicwater-soluble polymer can be direct, wherein no intervening atoms arelocated between the anti-TNF antibody and the polymer, or indirect,wherein one or more atoms are located between the anti-TNF antibody andthe polymer. With respect to the indirect attachment, a “spacer moiety”serves as a linker between the residue of anti-TNFα antibody and thewater-soluble polymer. The one or more atoms making up the spacer moietycan include one or more of carbon atoms, nitrogen atoms, sulfur atoms,oxygen atoms, and combinations thereof. The spacer moiety can comprisean amide, secondary amine, carbamate, thioether, and/or disulfide group.Nonlimiting examples of specific spacer moieties include those selectedfrom the group consisting of —O—, —S—, —S—S—, —C(O)—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—, bivalent cycloalkyl group, —O—,—S—, an amino acid, —N(R⁶)—, and combinations of two or more of any ofthe foregoing, wherein R⁶ is H or an organic radical selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) iszero to six, and (j) is zero to 20. Other specific spacer moieties havethe following structures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, whereinthe subscript values following each methylene indicate the number ofmethylenes contained in the structure, e.g., (CH₂)₁₋₆ means that thestructure can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, anyof the above spacer moieties may further include an ethylene oxideoligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e.,—(CH₂CH₂O)₁₋₂₀]. That is, the ethylene oxide oligomer chain can occurbefore or after the spacer moiety, and optionally in between any twoatoms of a spacer moiety comprised of two or more atoms. Also, theoligomer chain would not be considered part of the spacer moiety if theoligomer is adjacent to a polymer segment and merely represent anextension of the polymer segment.

Compositions

The conjugates are typically part of a composition. Generally, thecomposition comprises a plurality of conjugates, preferably although notnecessarily, each conjugate is comprised of the same anti-TNFα antibody(i.e., within the entire composition, only one type of anti-TNFαantibody is found). In addition, the composition can comprise aplurality of conjugates wherein any given conjugate is comprised of amoiety selected from the group consisting of two or more differentanti-TNFα antibodies (i.e., within the entire composition, two or moredifferent anti-TNFα antibodies are found). Optimally, however,substantially all conjugates in the composition (e.g., 85% or more ofthe plurality of conjugates in the composition) are each comprised ofthe same anti-TNFα antibody.

The composition can comprise a single conjugate species (e.g., amonoPEGylated conjugate wherein the single polymer is attached at thesame location for substantially all conjugates in the composition) or amixture of conjugate species (e.g., a mixture of monoPEGylatedconjugates where attachment of the polymer occurs at different sitesand/or a mixture monPEGylated, diPEGylated and triPEGylated conjugates).The compositions can also comprise other conjugates having four, five,six, seven, eight or more polymers attached to any given moiety havinganti-TNFα antibody activity. In addition, the invention includesinstances wherein the composition comprises a plurality of conjugates,each conjugate comprising one water-soluble polymer covalently attachedto one anti-TNFα antibody, as well as compositions comprising two,three, four, five, six, seven, eight, or more water-soluble polymerscovalently attached to one anti-TNFα antibody.

With respect to the conjugates in the composition, the composition willsatisfy one or more of the following characteristics: at least about 85%of the conjugates in the composition will have from one to four polymersattached to the anti-TNFα antibody; at least about 85% of the conjugatesin the composition will have from one to four polymers attached to theanti-TNFα antibody moiety; at least about 85% of the conjugates in thecomposition will have from one to three polymers attached to theanti-TNFα antibody moiety; at least about 85% of the conjugates in thecomposition will have from one to two polymers attached to the anti-TNFαantibody; at least about 85% of the conjugates in the composition willhave one polymer attached to the anti-TNFα antibody moiety; at leastabout 95% of the conjugates in the composition will have from one tofive polymers attached to the anti-TNFα antibody moiety; at least about95% of the conjugates in the composition will have from one to fourpolymers attached to the anti-TNFα antibody; at least about 95% of theconjugates in the composition will have from one to three polymersattached to the anti-TNFα antibody moiety; at least about 95% of theconjugates in the composition will have from one to two polymersattached to the anti-TNFα antibody; at least about 95% of the conjugatesin the composition will have one polymer attached to the anti-TNFαantibody; at least about 99% of the conjugates in the composition willhave from one to five polymers attached to the anti-TNFα antibodymoiety; at least about 99% of the conjugates in the composition willhave from one to four polymers attached to the anti-TNFα antibody; atleast about 99% of the conjugates in the composition will have from oneto three polymers attached to the anti-TNFα antibody; at least about 99%of the conjugates in the composition will have from one to two polymersattached to the anti-TNFα antibody; and at least about 99% of theconjugates in the composition will have one polymer attached to theanti-TNFα antibody.

In one or more embodiments, it is preferred that theconjugate-containing composition is free or substantially free ofalbumin. It is also preferred that the composition is free orsubstantially free of proteins that do not have anti-TNFα antibody.Thus, it is preferred that the composition is 85%, more preferably 95%,and most preferably 99% free of albumin. Additionally, it is preferredthat the composition is 85%, more preferably 95%, and most preferably99% free of any protein that does not have anti-TNF antibody activity.To the extent that albumin is present in the composition, exemplarycompositions of the invention are substantially free of conjugatescomprising a poly(ethylene glycol) polymer linking a residue of ananti-TNFα antibody to albumin.

Control of the desired number of polymers for any given moiety can beachieved by selecting the proper polymeric reagent, the ratio ofpolymeric reagent to the anti-TNFα antibody moiety, temperature, pHconditions, and other aspects of the conjugation reaction. In addition,reduction or elimination of the undesired conjugates (e.g., thoseconjugates having four or more attached polymers) can be achievedthrough purification means.

For example, the polymer-anti-TNFα antibody conjugates can be purifiedto obtain/isolate different conjugated species. Specifically, theproduct mixture can be purified to obtain an average of anywhere fromone, two, three, four, five or more PEGs per anti-TNF antibody,typically one, two or three PEGs per anti-TNFα antibody. The strategyfor purification of the final conjugate reaction mixture will dependupon a number of factors, including, for example, the molecular weightof the polymeric reagent employed, the particular anti-TNFα antibody,the desired dosing regimen, and the residual activity and in vivoproperties of the individual conjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography and/or ion exchangechromatography. That is to say, gel filtration chromatography is used tofractionate differently numbered polymer-to-anti-TNFα antibody ratios(e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates 1polymer to anti-TNFα antibody, “2-mer” indicates two polymers toanti-TNFα antibody moiety, and so on) on the basis of their differingmolecular weights (where the difference corresponds essentially to theaverage molecular weight of the water-soluble polymer portion). Forexample, in an exemplary reaction where a 35,000 Dalton protein israndomly conjugated to a polymeric reagent having a molecular weight ofabout 20,000 Daltons, the resulting reaction mixture may containunmodified protein (having a molecular weight of about 35,000 Daltons),monoPEGylated protein (having a molecular weight of about 55,000Daltons), diPEGylated protein (having a molecular weight of about 75,000Daltons), and so forth.

While this approach can be used to separate PEG and otherpolymer-anti-TNFα antibody moiety conjugates having different molecularweights, this approach is generally ineffective for separatingpositional isoforms having different polymer attachment sites within theanti-TNFα antibody moiety. For example, gel filtration chromatographycan be used to separate from each other mixtures of PEG 1-mers, 2-mers,3-mers, and so forth, although each of the recovered conjugatecompositions may contain PEG(s) attached to different reactive groups(e.g., lysine residues) within the anti-TNFα antibody.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) absorbance at 280 nm forprotein content, (ii) dye-based protein analysis using bovine serumalbumin (BSA) as a standard, (iii) iodine testing for PEG content (Simset al. (1980) Anal. Biochem, 107:60-63), (iv) sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS AGE), followed by staining withbarium iodide, and (v) high performance liquid chromatography (HPLC).

Separation of positional isoforms is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) using a suitable column (e.g., a C18 column orC3 column, available commercially from companies such as AmershamBiosciences or Vydac) or by ion exchange chromatography using an ionexchange column, e.g., a Sepharose™ ion exchange column available fromAmersham Biosciences. Either approach can be used to separatepolymer-active agent isomers having the same molecular weight (i.e.,positional isoforms).

The compositions are preferably substantially free of proteins that donot have anti-TNFα antibody activity. In addition, the compositionspreferably are substantially free of all other noncovalently attachedwater-soluble polymers. In some circumstances, however, the compositioncan contain a mixture of polymer-anti-TNFα antibody conjugates andunconjugated anti-TNFα antibody.

Optionally, the composition of the invention further comprises apharmaceutically acceptable excipient. If desired, the pharmaceuticallyacceptable excipient can be added to a conjugate to form a composition.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The composition can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for one or more embodiments of the present inventioninclude benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the composition as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in one or more embodiments of the present inventioninclude, for example, ascorbyl palmitate, butylated hydroxyanisole,butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propylgallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite, and combinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases can be present as an excipient in the composition.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate (i.e., the conjugate formed between theactive agent and the polymeric reagent) in the composition will varydepending on a number of factors, but will optimally be atherapeutically effective dose when the composition is stored in a unitdose container (e.g., a vial). In addition, the pharmaceuticalpreparation can be housed in a syringe. A therapeutically effective dosecan be determined experimentally by repeated administration ofincreasing amounts of the conjugate in order to determine which amountproduces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about 5%to about 98% by weight, more preferably from about 15 to about 95% byweight of the excipient, with concentrations less than 30% by weightmost preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The compositions encompass all types of formulations and in particularthose that are suited for injection, e.g., powders or lyophilates thatcan be reconstituted as well as liquids. Examples of suitable diluentsfor reconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.

The compositions of one or more embodiments of the present invention aretypically, although not necessarily, administered via injection and aretherefore generally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering to a patient, generally via injection, a therapeuticallyeffective amount of the conjugate (preferably provided as part of apharmaceutical composition). As previously described, the conjugates canbe administered parenterally by intravenous injection. Advantageously,the conjugate can also be administered by intramuscular or bysubcutaneous injection. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the conjugate. Those ofordinary skill in the art appreciate which conditions a specificconjugate can effectively treat. For example, the conjugates can be usedeither alone or in combination with other pharmacotherapy to treatpatients suffering arthritis, Crohn's disease, psoriatic arthritis,ulcerative colitis, plaque psoriasis, sarcoidosis, ankylosingspondylitis, and cytokine-induced islet destruction in automimmunediabetes. Advantageously, the conjugate can be administered to thepatient prior to, simultaneously with, or after administration ofanother active agent.

The actual dose to be administered will vary depending upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 100mg, preferably in doses from 0.01 mg/day to 75 mg/day, and morepreferably in doses from 0.10 mg/day to 50 mg/day. A given dose can beperiodically administered up until, for example, symptoms of arthritislessen and/or are eliminated entirely.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis, biochemistry, proteinpurification and the like, which are within the skill of the art. Suchtechniques are fully explained in the literature. See, for example, J.March, Advanced Organic Chemistry: Reactions Mechanisms and Structure,4th Ed. (New York: Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be taken into account.Unless indicated otherwise, temperature is in degrees C. and pressure isat or near atmospheric pressure at sea level. Each of the followingexamples is considered to be instructive to one of ordinary skill in theart for carrying out one or more of the embodiments described herein.

Infliximab was purchased commercially from a pharmaceutical distributoras a lyophilized powder and was reconstituted immediately prior to usewith sterile water to yield a reconstituted stock infliximab liquid at aconcentration of 10 mg/mL and thereafter stored at 4° C.

SDS-PAGE Analysis

In some instances, samples were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using anInvitrogen system (SureLock II Precast Gel Electrophoresis System).Samples were mixed with sample buffer. Then, the prepared samples wereloaded onto a gel and ran for approximately thirty minutes.

Anion Exchange Chromatography

In some instances, a Hitrap Q Sepharose HP anion exchange column (5 ml,Amersham Biosciences) was used with the AKTAprime system (AmershamBiosciences) to purify the prepared conjugates. For each conjugatesolution prepared, the conjugate was loaded on a column that ispre-equilibrated in 50 mM MES buffer, pH 5.4 (buffer A) and is thenwashed with nine column volumes of buffer A to remove any unreacted PEGreagent. Subsequently, a gradient of buffer A with 0-100% buffer B (50mM MES with 0.5 M NaCl buffer, pH 5.4) was raised. The eluent wasmonitored by UV detector at 280 nm. Any higher-mers (e.g., 11-mers,10-mers, and so forth) will elute first, followed by increasinglysmaller and smaller conjugates (e.g, 5-mers and 4-mers, and so forth),until 1-mers, and finally, unconjugated infliximab species elute. Thefractions can be pooled and the purity of the individual conjugate wasdetermined by SEC-HPLC mostly by SDS-PAGE.

SEC-HPLC Analysis

In some instances, size exclusion chromatography (SEC-HPLC) analysis wasperformed on an Agilent 1100 HPLC system (Agilent). Samples are analyzedusing a GF-450 Zorbax (Agilent), and a mobile phase consisting of 90%phosphate buffered saline and 10% ethanol, pH 7.2. The flow rate for thecolumn can be 0.5 ml/min. Eluted protein and PEG-protein conjugates canbe detected using UV at 280 nm.

Example 1 PEGylation of Infliximab with mPEG-SPA, 30 kDa (10:1 Polymerto Infliximab Ratio)

mPEG-SPA, 30 kDa

MPEG-SPA, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. A ten-fold excess (relative to the amount of infliximab ina measured aliquot of the stock infliximab liquid) of the warmedmPEG-SPA was dissolved in buffer 2 mM HCL (<10% the reaction volume) andis added to an aliquot of the stock infliximab liquid and mixed well.After the addition of the mPEG-SPA, the pH of the reaction mixture isdetermined and adjusted to pH 7.2-7.5 using conventional techniques. Toallow for coupling of the mPEG-SPA to infliximab via an amide linkage,the reaction solution is stirred for five hours at room temperature andthereafter is stirred overnight at 3-8° C. in a cold room in the dark,thereby resulting in a conjugate solution. The reaction was quenchedwith glycine.

According to SDS-PAGE analysis, approximately 45% PEGylation occurred,which consisted mostly of 1-mers, 2-mers, 3-mers and some 4-mers.

Using this same approach, other conjugates can be prepared usingmPEG-SPA having other weight average molecular weights.

Example 2

Example 1 was repeated and served as a control for Example 2b

Example 2b PEGylation of Infliximab with in PEG-SPA, 30 kDa (10:1Polymer to Infliximab Ratio, with Blocking Agent)

mPEG-SPA, 30 kDa

Prior to the conjugation reaction, 0.164 mg of infliximab in sterilewater was obtained and pH tested and adjusted to 8.0. To reversiblyprotect the most reactive amino groups in infliximab, this liquid wascombined with a 10-fold molar excess of dimethylmaleic anhydride,“DMMAn”, (Tsunoda, S., et al., J. Pharmacol. Exp. Ther. 1999, 290,368-72) relative to moles of infliximab, to thereby form a DMMAn-treatedinfliximab liquid. The protection reactions were allowed to proceed for30 minutes at 37° C. The pH was tested and adjusted as necessary toensure a pH of 8.0.

MPEG-SPA, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. The mPEG-SPA, 30 kDa, (1.4 mg) was dissolved in 11.6 μL of2 mM HCl to form an mPEG-SPA solution. The mPEG-SPA solution was addedto the DMMAn-treated infliximab liquid (pH 8.0, room temperature), untila ten-fold molar excess of mPEG-SPA relative to infliximab was reached.To allow for coupling of the mPEG-SPA to infliximab via an amidelinkage, the reaction solution was stirred for two hours at roomtemperature and then overnight (16 hours) at 6° C., thereby resulting ina conjugate solution. The reaction was quenched by addition of glycine.Thereafter, to deprotect the protected lysine amino groups, the reactionmixture was adjusted to pH 6.0 with 0.1 N HCl and incubated at 37° C.for 30 minutes.

According to SDS-PAGE analysis, approximate 20% monoPEGylation occurred.

Using this same approach, other conjugates can be prepared usingmPEG-SPA having other weight average molecular weights.

Example 3a PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 60 kDa (10:1 Polymer to Infliximab Ratio)

Branched mPEG2-N-Hydroxysuccinimide, 60 kDa

Branched mPEG2-N-hydroxysuccinimide, 60 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide (0.326 mg) was dissolved in 32.6 μL of 2 mMHCl to form a branched mPEG2-N-hydroxysuccinimide solution. The branchedmPEG2-N-hydroxysuccinimide solution was added to a previously preparedinfliximab reaction mixture of 164 μg until a ten-fold molar excess ofthe branched mPEG2-N-hydroxysuccinimide to infliximab was reached. ThepH was tested and adjusted as necessary to ensure a pH of 8.0. To allowfor coupling of the branched mPEG2-N-hydroxysuccinimide to infliximabvia an amide linkage, the reaction solution was stirred for two hours atroom temperature and then overnight (sixteen hours) at 6° C., therebyresulting in a conjugate solution. The reaction was quenched by additionof glycine and the pH was reduced to a pH of 6.0.

According to SDS-PAGE analysis, approximately 40% of the native wasconjugated to PEG. The reaction yielded mostly 1-mers and some 2-mersand 3-mers.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 3b PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 60 kDa (10:1 Polymer to Infliximab Ratio,with Blocking Agent)

Branched mPEG2-N-Hydroxysuccinimide, 60 kDa

Prior to the conjugation reaction, 0.164 mg of infliximab in sterilewater was obtained and pH tested and adjusted to 8.0. To reversiblyprotect the most reactive amino groups in infliximab, this liquid wascombined with a ten-fold molar excess of dimethylmaleic anhydride,“DMMAn”, (Tsunoda, S., et al., J. Pharmacol. Exp. Ther. 1999, 290,368-72) relative to moles of infliximab, to thereby form a DMMAn-treatedinfliximab liquid. The protection reactions were allowed to proceed for30 minutes at 37° C. The pH was tested and adjusted as necessary toensure a pH of 8.0.

Branched mPEG2-N-hydroxysuccinimide, 60 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide, 60 kDa, (0.326 mg) was dissolved in 32.6 μLof 2 mM HCl to form a branched mPEG2-N-hydroxysuccinimide solution. Thebranched mPEG2-N-hydroxysuccinimide solution was added to theDMMAn-treated infliximab liquid (pH 8.0, room temperature), until aten-fold molar excess of branched mPEG2-N-hydroxysuccinimide relative toinfliximab was reached. To allow for coupling of the branchedmPEG2-N-hydroxysuccinimide to infliximab via an amide linkage, thereaction solution was stirred for two hours at room temperature andthereafter stirred overnight (sixteen hours) at 6° C., thereby resultingin a conjugate solution. The reaction was quenched by addition ofglycine. Thereafter, to deprotect the protected lysine amino groups, thereaction mixture was adjusted to pH 6.0 with 0.1 N HCl and incubated at37° C. for 30 minutes.

According to SDS-PAGE analysis, approximately 20% of the nativeinfliximab was conjugate to PEG. The reaction yield mostly 1-mers andsome 2-mers.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 4a (Unreduced) PEGylation of Infliximab with mPEG-MAL, 30 kDa(10:1 Polymer to Infliximab Ratio)

MPEG-MAL, 30 kDa

This reaction was to use as a control for Example 4b. By not reducingthe infliximab, it was possible to identify whether there were any freethiol groups associated with the side chains of cysteine residues.

Prior to the conjugation reaction, 0.164 mg of infliximab in sterilewater was obtained. To this was added mPEG-MAL, 30 kDa, (previouslystored at −20° C. under argon, warmed to ambient temperature, 0.328 mgof which was dissolved in 0.1 mL of 2 mM HCl) until a ten-fold molarexcess of mPEG-MAL to infliximab was reached. To allow for conjugation,the mixture was stirred at room temperature for three hours.

According to SDS-PAGE analysis, less than 10% of 1-mer conjugates weredetected.

Using this same approach, other conjugates can be prepared usingmPEG-MAL having other weight average molecular weights.

Example 4b PEGylation of Infliximab with mPEG-MAL, 30 kDa [10:1 Polymerto Infliximab Ratio, DTT (Reducing Agent)]

mPEG-MAL, 30 kDa

In most cases, reducing an antibody will create a fragment that cannotbe returned to a similar conformation after PEGylation. In any event,this reaction was performed to compare the number of free cysteineresidues prior to reduction to the number of free cysteine residuesfollowing reduction.

Prior to the conjugation reaction, 0.164 mg of infliximab in sterilewater was obtained. To this was added 1.68 mg of DTT (in a ten-foldexcess relative to moles of infliximab) to allow for reduction at roomtemperature for one hour. The DTT was then removed via DeSalt media andconcentrated back down to ˜1 mL of stock solution with a 30K Mw cutoffmembrane to form a reduced stock infliximab liquid.

MPEG-MAL, 30 kDa, stored at −20° C. under argon was warmed to ambienttemperature. The warmed mPEG-MAL (0.328 mg) was dissolved in 0.1 mL of 2mM HCl to form an mPEG-MAL solution. To the reduced stock infliximabliquid was added the mPEG-MAL solution to result in a ten-fold molarexcess of mPEG-MAL. The mixture was stirred at room temperature forthree hours.

According to SDS-PAGE analysis, 1-mers (about 20%) and 2-mers (about 5%)were detected.

Using this same approach, other conjugates can be prepared usingmPEG-MAL having other weight average molecular weights.

Example 5 PEGylation of Infliximab with mPEG-SMB, 30 kDa (200:1 Polymerto Infliximab Ratio)

mPEG-SMB, 30 kDa

mPEG-SMB, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed MPEG-SMB (6.56 mg) was dissolved in 0.2 mL of 2mM HCl to form an mPEG-SMB solution. The MPEG-SMB solution was added toan aliquot of the stock infliximab liquid containing 0.164 mg ofinfliximab until a 200 molar excess of mPEG-SMB relative to infliximabwas reached. After the addition of the mPEG-SMB, the pH of the reactionmixture was tested to ensure a pH of 7.2 to 7.5. To allow for couplingof the mPEG-SMB to infliximab via an amide linkage, the reactionsolution was stirred for three hours at room temperature. Coupling wasallowed to continue by stirring the reaction solution overnight (sixteenhours) at 6° C., thereby resulting in a conjugate solution. The reactionwas quenched with glycine.

According to SDS-PAGE analysis, 1-mers (about 40%/O) and 2-mers, 3-mersand 4-mers (totaling about 20%) were detected.

Using this same approach, other conjugates can be prepared usingmPEG-SMB having other weight-average molecular weights.

Example 6 Selective N-Terminal PEGylation of Infliximab with LinearmPEG-Butyraldehyde, 30 kDa (200:1 Polymer to Infliximab Ratio)

Linear mPEG-Butyraldehyde Derivative, 30 kDa (“mPEG-ButyrALD”)

mPEG-Butyraldehyde, 30 kDa, stored at −20° C. under argon, was warmed toambient temperature. The mPEG-butyraldehyde (6.56 mg) was dissolved in0.2 mL of 2 mM HCl to form an mPEG-butyraldehyde solution. ThemPEG-butyraldehyde solution was added to a previously preparedinfliximab reaction mixture (0.164 mg stock infliximab liquid, pHadjusted to 6.0 via conventional methods) until a 200 molar excess ofmPEG-butryaldehyde to infliximab was reached. After addition of themPEG-butyraldehyde, the pH was tested and adjusted as necessary toensure a pH of about 6.0. A reducing agent, NaCNBH₃, was added at afive-fold molar excess relative to the branched mPEG-butyraldehyde (withthe pH tested and adjusted as necessary to ensure a pH of about 6.0).The solution was then stirred for two hours at room temperature and thenovernight at 4° C. to ensure coupling via an amine linkage.

According to SDS-PAGE analysis, 1-mers (about 40%) and 2-mers and 3-mers(totaling about 10%) were detected.

According to SDS-PAGE analysis, 1-mers (about 40%) and 2-mers and 3-mers(totaling about 10%) were detected. It is noted that N-terminalPEGylation could decrease the conjugate's ability to find to TNFα;binding activity assays for the resulting conjugate are particularlywarranted.

Using this same approach, other conjugates can be prepared usingmPEG-butyraldehyde having other weight average molecular weights.

Example 7 PEGylation of Infliximab with mPEG-PIP, 20 kDa (200:1 Polymerto Infliximab Ratio)

mPEG-PIP, 20 kDa (ketone and acetal forms)

MPEG-PIP, 20 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. The mPEG-PIP (4.4 mg) was dissolved in 0.2 mL of 2 mM HClto form an mPEG-PIP solution. The MPEG-PIP solution was added to apreviously prepared infliximab reaction mixture (0.164 mg stockinfliximab liquid, pH adjusted to 6.0 via conventional methods) until a200 molar excess of mPEG-PIP to infliximab was reached. After additionof the mPEG-PIP, the pH was tested and adjusted as necessary to ensure apH of about 6.0. A reducing agent, NaCNBH₃, was added at a five-foldmolar excess relative to the mPEG-PIP (with the pH tested and adjustedas necessary to ensure a pH of about 6.0). The solution was then stirredfor two hours at room temperature and then overnight at 4° C. to ensurecoupling via an amine linkage.

According to SDS-PAGE analysis, about 10% of 1-mer conjugates weredetected.

Example 8a PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 40 kDa (200:1 Polymer to Infliximab Ratio)

Branched mPEG2-N-Hydroxysuccinimide, 40 kDa

Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide (8.7 mg) was dissolved in 200 μL of 2 mM HClto form a branched mPEG2-N-hydroxysuccinimide solution. The branchedmPEG2-N-hydroxysuccinimide solution was added to a previously preparedinfliximab reaction mixture (0.164 mg stock infliximab liquid, pH 8.0)until a 200-fold molar excess of the branched mPEG2-N-hydroxysuccinimideto infliximab was reached. The pH was tested and adjusted as necessaryto ensure a pH of 8.0. To allow for coupling of the branchedmPEG2-N-hydroxysuccinimide to infliximab via an amide linkage, thereaction solution was stirred for two hours at room temperature and thenovernight (sixteen hours) at 6° C., thereby resulting in a conjugatesolution. The pH was reduced to 6.0 with 0.1 M HCl to release theanhydride. The reaction was quenched by addition of glycine. Accordingto SDS-PAGE analysis, about 10% of 1-mer conjugates were detected.

According to SDS-PAGE analysis, about 30% of 1-mer conjugates weredetected.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 8b PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 40 kDa (200:1 Polymer to Infliximab Ratio,with Blocking Agent)

Branched mPEG2-N-Hydroxysuccinimide, 40 kDa

Prior to the conjugation reaction, 0.164 mg of infliximab in sterilewater was obtained and pH tested and adjusted to 8.0. To reversiblyprotect the most reactive amino groups in infliximab, this liquid wascombined with a ten-fold molar excess of dimethylmaleic anhydride,“DMMAn”, (13.8 mg) (Tsunoda, S., et al., J. Pharmacol. Exp. Ther. 1999,290, 368-72) relative to moles of infliximab, to thereby form aDMMAn-treated infliximab liquid. The protection reactions were allowedto proceed for 30 minutes at 37° C. The pH was tested and adjusted asnecessary to ensure a pH of 8.0.

Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide, 40 kDa, (8.7 mg) was dissolved in 32.6 μL of2 mM HCl to form a branched mPEG2-N-hydroxysuccinimide solution. Thebranched mPEG2-N-hydroxysuccinimide solution was added to theDMMAn-treated infliximab liquid (pH 8.0, room temperature), until a200-fold molar excess of branched mPEG2-N-hydroxysuccinimide relative toinfliximab was reached. To allow for coupling of the branchedmPEG2-N-hydroxysuccinimide to infliximab via an amide linkage, thereaction solution was stirred for two hours at room temperature andthereafter stirred overnight (sixteen hours) at 6° C., thereby resultingin a conjugate solution. The reaction was quenched by addition ofglycine. Thereafter, to deprotect the protected lysine amino groups, thereaction mixture was adjusted to pH 6.0 with 0.1 N HCl and incubated at37° C. for 30 minutes.

According to SDS-PAGE analysis, approximately 10% of 1-mers weredetected.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 9 Scale Up of Example 5

mPEG-SMB, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed mPEG-SMB (65.6 mg) was dissolved in 2.0 mL of 2mM HCl to form an mPEG-SMB solution. The mPEG-SMB solution was added toan aliquot of the stock infliximab suspension containing 1.64 mg ofinfliximab until a 200 molar excess of nipEG-SMB relative to infliximabwas reached. After the addition of the mPEG-SMB, the pH of the reactionmixture was tested to ensure a pH of 7.2 to 7.5. To allow for couplingof the mPEG-SMB to infliximab via an amide linkage, the reactionsolution was stirred for three hours at room temperature. Coupling wasallowed to continue by stirring the reaction solution overnight (sixteenhours) at 6° C., thereby resulting in a conjugate solution. The reactionwas quenched with glycine.

According to SDS-PAGE analysis, 1-mers (about 40%) and 2-mers and 3-mers(totaling about 20%) were detected.

Example 10 Scale Up of Example 8A

Example 8A was carried out again, but on larger scale. The results andyield were similar.

Example 11 PEGylation of Infliximab with mPEG-SPA, 5 kDa (20:1 Polymerto Infliximab Ratio)

mPEG-SPA, 5 kDa

mPEG-SPA, 5 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. A twenty-fold excess (relative to the amount of infliximabin a measured aliquot of the stock infliximab suspension) of the warmedMPEG-SPA was dissolved in buffer (164 μl of 1 mg/mL PEG solution in 2 mMHCl) and is added to an aliquot of the stock infliximab liquid (0.246 mginfliximab) and mixed well. After the addition of the mPEG-SPA, the pHof the reaction mixture is determined and adjusted to 7.2-7.5 usingconventional techniques. To allow for coupling of the MPEG-SPA toinfliximab via an amide linkage, the reaction solution is stirred forfive hours at room temperature in the dark and thereafter is stirredovernight at 3-8° C. in a cold room in the dark, thereby resulting in aconjugate solution. The reaction was quenched with glycine.

PEGylation yields were not determined given the relatively smalldifference in change of molecular weights between the unconjugated andconjugated forms.

Using this same approach, other conjugates are prepared using mPEG-SPAhaving other weight average molecular weights.

Example 12 PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 60 kDa (20:1 Polymer to Infliximab Ratio)

Branched mPEG2-N-Hydroxysuccinimide, 60 kDa

Branched mPEG2-N-hydroxysuccinimide, 60 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide (0.650 mg) was dissolved in 32.6 μL of 2 mMHCl to form a branched mPEG2-N-hydroxysuccinimide solution. The branchedmPEG2-N-hydroxysuccinimide solution was added to a previously preparedinfliximab reaction mixture of 164 μg (raising the pH to 7.0 byconventional methods) until a ten-fold molar excess of the branchedmPEG2-N-hydroxysuccinimide to infliximab was reached. The pH was testedand adjusted as necessary to ensure a pH of 7.0. To allow for couplingof the branched mPEG2-N-hydroxysuccinimide to infliximab via an amidelinkage, the reaction solution was stirred for two hours at roomtemperature and then overnight (sixteen hours) at 6° C., therebyresulting in a conjugate solution. The reaction was quenched by additionof glycine.

According to SDS-PAGE analysis, approximately 20% of the native wasconjugated to PEG. The reaction yielded mostly 1-mers and some 2-mers.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 13 PEGylation of Infliximab with a branched mPEG-MAL, 60 kDa(“mPEG2-MAL”) (20:1 Polymer to Infliximab Ratio)

In most cases, reducing an antibody will create a fragment that cannotbe returned to a similar conformation after PEGylation. In any eventthis reaction was performed to compare the number of free cysteineresidues prior to reduction to the number of free cysteine residuesfollowing reduction.

Prior to the conjugation reaction, 01.64 mg of infliximab in sterilewater was obtained. To this was added 1.68 mg of DTT (in a ten foldexcess relative to moles of infliximab) to allow for reduction at roomtemperature for one hour. The DTT was then removed via DeSalt media andconcentrated back down to ˜1 mL of stock solution with a 30K Mw cutoffmembrane to form a reduced stock infliximab liquid.

Branched mPEG-MAL, 60 kDa, stored at −20° C. argon was warmed to ambienttemperature. The warmed branched mPEG-MAL (0.652 μg) was dissolved in0.1 mL of 2 mM HCl to form a branched mPEG-MAL solution. To a reducedstock infliximab liquid was added the branched mPEG-MAL solution toresult in a twenty-fold molar excess of mPEG-MAL. The mixture wasstirred at room temperature for three hours.

According to SDS-PAGE analysis, 1-mers (about 20%) were formed.

Using this same approach, other conjugates can be prepared usingmPEG-MAL having other weight average molecular weights.

Example 14 PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 60 kDa (Two×20:1 Polymer to InfliximabRatio)

Branched mPEG2-N-Hydroxysuccinimide, 60 kDa

Branched mPEG2-N-hydroxysuccinimide, 60 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide (0.650 mg) was dissolved in 32.6 μL of 2 mMHCl to form a branched mPEG2-N-hydroxysuccinimide solution. The branchedmPEG2-N-hydroxysuccinimide solution was added to an aliquot of the stockinfliximab liquid (164 μg of infliximab)(stock infliximab liquid pHadjusted to 7.0 using conventional methods) until a twenty-fold molarexcess of the branched mPEG2-N-hydroxysuccinimide to infliximab wasreached. The pH was tested and adjusted as necessary to ensure a pH of7.0. The solution reacted for 30 minutes and then a second addition ofmPEG2-N-hydroxysuccinimide solution was prepared and added in the samemanner as before. To allow for coupling of the branchedmPEG2-N-hydroxysuccinimide to infliximab via an amide linkage, thereaction solution was stirred for two hours at room temperature and thenovernight (sixteen hours) at 6° C., thereby resulting in a conjugatesolution. The reaction was quenched by addition of glycine.

According to SDS-PAGE analysis, approximately 30% of the native wasconjugated to PEG. The reaction yielded mostly 1-mers and some 2-mers.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 15 PEGylation of Infliximab with BranchedmPEG2-N-Hydroxysuccinimide, 60 kDa (Two×10):1 Polymer to InfliximabRatio)

Branched mPEG2-N-Hydroxysuccinimide, 60 kDa

Branched mPEG2-N-hydroxysuccinimide, 60 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide (0.326 mg) was dissolved in 32.6 μL of 2 mMHCl to form a branched mPEG2-N-hydroxysuccinimide solution. The branchedmPEG2-N-hydroxysuccinimide solution was added to an aliquot of the stockinfliximab liquid (164 μg of infliximab)(stock infliximab liquid pHadjusted to 7.0 using conventionals methods) until a ten-fold excess ofthe branched mPEG2-N-hydroxysuccinimide to infliximab was reached. ThepH was tested and adjusted as necessary to ensure a pH of 7.0. Thesolution reacted for 30 minutes and then a second addition ofmPEG2-N-hydroxysuccinimide solution was prepared and added in the samemanner as before. To allow for coupling of the branchedmPEG2-N-hydroxysuccinimide to infliximab via an amide linkage, thereaction solution was stirred for two hours at room temperature and thenovernight (sixteen hours) at 6° C., thereby resulting in a conjugatesolution. The reaction was quenched by addition of glycine.

According to SDS-PAGE analysis, approximately 30% of the native wasconjugated to PEG. The reaction yielded mostly 1-mers and some 2-mers.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 16

Conjugates prepared in accordance with the Examples and precedingdescription were tested for activity based on a radioligand bindingassay. The following materials were used: source, Human U937 cells;ligand, 0.028 nM [¹²⁵I] TNF-α; vehicle, 1% 50 mM NaPO₄ pH 8.0;incubation time/temperature, three hours at 4° C.; incubation buffer, 50mM Tris-HCl, pH 7.4, 0.5 mM EDTA at 4° C.; non-specific ligand, 0.04 μMTNF-α; K_(D), 0.07 nM; B_(MAX), 0.2 pmole/mg protein; specific binding,60%; quantitation method, radioligand binding; significance criteria,≧50% of max stimulation or inhibition. Where presented, IC₅₀ values weredetermined by a non-linear, least squares regression analysis using DataAnalysis Toolbox™ (MDL Information Systems, San Leandro, Calif.). Whereinhibition constants (K_(I)) are presented, the K₁ values are calculatedusing the equation of Cheng and Prusoff (Cheng et al. Biochem.Pharmacol. 22:3099-3108, 1973) using the observed IC₅₀ of the testedcompound, the concentration of radioligand employed in the assay, andthe historical values for the K_(D) of the ligand (obtainedexperimentally). Where presented, the Hill coefficient (n_(H)), definingthe sloop of the competitive binding curve, was calculated using DataAnalysis ToolboX™. Hill coefficients significantly different than 1.0,may suggest that binding displacement does not follow the laws of massaction with a single binding site. Where IC₅₀, K_(I), and/or n_(H) dataare presented without Standard Error of the Mean (SEM), data areinsufficient to be quantitative, and the values presented (IC₅₀, K_(I),n_(H)) should be interpreted with caution. Results are provided in Table4.

TABLE 4 Activity Based on a Radioligand Binding Assay Compound % IDConcentration Inhibition IC₅₀ K_(I) n_(H) Conjugate A 3 nM 51 2.85 ±0.204 nM  2.04 ± 0.145 nM 1.43 ± 0.162 Conjugate B 11 nM  52 0.850 ±0.063 nM  0.607 ± 0.045 nM 1.76 ± 0.165 Conjugate C 3 nM 62 1.54 ± 0.178nM  1.1 ± 0.127 nM 1.43 ± 0.098 Conjugate D 1 nM 58 0.636 ± 0.039 nM 0.454 ± 0.028 nM 1.59 ± 0.115 Conjugate E 10 nM  50 9.87 ± 0.782 nM 7.05 ± 0.558 nM 1.65 ± 0.162

What is claimed is:
 1. A conjugate comprising a single water-solublepolymer covalently attached, either directly or through a spacer moietyof one to twenty atoms, to an amino group of an amino acid side chain oran N-terminal amine of a residue of a full length anti-TNFα antibody,wherein: (i), the one to twenty atoms within the spacer moiety, whenpresent, correspond to the covalent attachment to the amino group of thefull length anti-TNFα antibody and atoms derived from a water-solublepolymeric reagent used to attach the water-soluble polymer; (ii) theanti-TNFα antibody is neither galactosylated nor glycosylated; (iii) thewater-soluble polymer is a polyethylene glycol terminally capped with amethoxy; and (iv) the water-soluble polymer has a weight-averagemolecular weight in the range of from about 10,000 Daltons to about100,000 Daltons.
 2. The conjugate of claim 1, having the followingstructure:

wherein: POLY is a water-soluble polymer; (a) is either zero or one; X,when present, is a spacer moiety comprised of one to twenty atoms; R¹ isH or an organic radical containing 1 to 3 carbon atoms; and ATA is aresidue of an anti-TNFα antibody.
 3. The conjugate of claim 1, havingthe following structure:

wherein: POLY is a water-soluble polymer; X, when present, is a spacermoiety comprised of one to twenty atoms; (a) is either zero or one; (j)is zero or an integer from 1 to about 20; (b) is zero or an integer from1 to about 10; each R², when present, is H or an organic radical; eachR³, when present, is H or an organic radical; and ATA is a residue of ananti-TNFα antibody.
 4. The conjugate of claim 1, wherein thewater-soluble polymer is branched.
 5. The conjugate of claim 1, whereinthe water-soluble polymer is linear.
 6. The conjugate of claim 1,wherein the anti-TNFα antibody is not a dimer or trimer.
 7. Theconjugate of claim 1, wherein the anti-TNFα antibody is monovalent. 8.The conjugate of claim 1, wherein the anti-TNFα antibody is notCDR-grafted.
 9. The conjugate of claim 2, having the followingstructure:

wherein n ranges from about 225 to about
 1930. 10. The conjugate ofclaim 2, having the following structure:

wherein n ranges from about 225 to about
 1930. 11. The conjugate ofclaim 1, wherein the poly(ethylene glycol) has a weight-averagemolecular weight in the range of from about 10,000 Daltons to about85,000 Daltons.
 12. The conjugate of claim 11, wherein the poly(ethyleneglycol) has a weight-average molecular weight in the range of from about20,000 Daltons to about 65,000 Daltons.
 13. The conjugate of claim 1,wherein the anti-TNFα antibody is either infliximab or adalimumab.
 14. Acomposition comprising the conjugate of claim 1 and a pharmaceuticallyacceptable excipient.